PASSING THE BATON IN A UBIQUITIN RELAY Regulated transfer of ubiquitin to protein substrates is required for normal neuronal function. The RING-Cys-Relay (RCR) ubiquitin E3 ligase (E3) uses a distinct mechanism involving the intramolecular relay of ubiquitin prior to its transfer to threonine residues [1]. A chemically engineered suicide substrate stabilises the highly transient ubiquitin transfer intermediate, allowing the visualisation of the initial handover of the ubiquitin baton to the RCR. The X-ray crystal structure of this transfer intermediate elucidates the RCR s novel mechanism.

STRUCTURAL BIOLOGY

50 ESRF

Structural base for the transfer of GPI- anchored glycoproteins into fungal cell walls, M.S. Vogt (a), G.F. Schmitz (b), D. Varón Silva (c), H.U. Mösch (b,d) and L.O. Essen (a,d), PNAS USA 117, 22061-22067 (2020); https://doi.

org/10.1073/pnas.2010661117. (a) Department of Chemistry, Philipps- Universität, Marburg (Germany) (b) Department of Biology, Philipps- Universität, Marburg (Germany) (c) Department of Biomolecular Systems,

Max Planck Institute of Colloids and Interfaces, Potsdam (Germany) (d) Center for Synthetic Microbiology, Philipps-Universität, Marburg (Germany)

[1] S. Sun et al., Curr. Biol. 30, R1163-R1169 (2020). [2] I. Delso et al., ChemMedChem 13, 128-132 (2018). [3] H. Kitagaki et al., Mol. Microbiol. 46, 1011-1022 (2002). [4] J. Ao et al., Eukaryot. Cell 14, 792-803 (2015). [5] F. Cuskin et al., Nature 517, 165-169 (2015). [6] A.J. Thompson et al., Angew. Chemie Int. Ed. 54, 5378-5382 (2015).

PRINCIPAL PUBLICATION AND AUTHORS

REFERENCES

competent binding mode of the GPI-core glycan within the binding pocket of Dfg5 does not allow modifications on mannose 1, which provides a rationale for using GPI-anchor modifications as a postcode to specifically sort proteins towards

their correct destination. These structural insights provide a basis for further structure- guided drug development by targeting Dfg5- like enzymes as elements of fungal cell wall biosynthesis.

MYCBP2 is a very large (~500 kDa) protein that regulates neurodevelopment and promotes programmed axon degeneration [2]. Previously, a 30 kDa region of MYCBP2 that contains the E3 machinery necessary for ubiquitin relay to threonine residues in substrate proteins was identified [1]. This machinery, and the unique mechanism it employs, was named RING-Cys- Relay (RCR). In order to ubiquitinate threonine substrates, the RCR receives ubiquitin from an E2 conjugating enzyme (E2) at an upstream

cysteine and subsequently relays the ubiquitin to a downstream cysteine. The downstream cysteine resides in a well-defined active site equipped with threonine esterification activity [1].

Numerous studies have suggested that the conformation adopted by the E2-Ub conjugate determines the reactivity and specificity of ubiquitin transfer. These studies have been limited to a small subset of the >600 human

Fig. 36: a) Transfer of ubiquitin to the upstream cysteine via transthiolation with E2-Ub. A transient tetrahedral intermediate is formed prior to intramolecular relay to the downstream cysteine. b) A semi-synthetic E2-ubiquitin conjugate acts as an ABP and chemically stabilises the otherwise transient transfer intermediate. The final three Ub residues are replaced by an ethyl linker, a triazole (Trz) group and a cysteine-reactive activated vinylsulfide (AVS) warhead.